(1) Field of the Invention
The present invention relates to active transfer of haptens, proteins, peptides, nucleic acids and other molecules into cells. More particularly, the present invention relates to novel polypeptides which can effectively penetrate into cells, in particular eukaryotic cells, and transport thereto a substance of interest which is capable of constituting novel antiviral compositions. This invention is of major importance as it has application in a variety of fields, in particular that of gene therapy and vaccines.
(2) Description of the Background
Gene therapy remains dependent on a considerable number of parameters, among them the development of vectors which are capable of transferring active principles endowed with predetermined specific properties to the cytoplasm of cells of the host organism under consideration in the absence of genetic alterations associated with the use of such vectors, and with no degradation of the biological activity of the transferred active principles. Current knowledge is that in spite of the effort achieved in developing vectors of viral or non viral origins, not all of these conditions have been satisfactorily fulfilled.
Further, the possibility of transporting substances efficiently into cells is also important for all biotechnological applications. Thus transferring substances into cells in vitro or ex vivo can be used either to produce proteins or peptides, or to regulate gene expression, or to analyse the properties of a given substance in that cell. In vivo, the transfer of a substance to a cell can also act to create models for studying diseases in animals or for studying the effect of a given compound on an organism.
The present invention thus aims to provide a novel type of vector which is both effective and is more innocuous than viral vectors in current use.
International patent application WO 97/02840 describes the use of antibodies or their F(ab′)2 and Fab′ fragments which can penetrate into the interior of living cells, as immunovectors for intracytoplasmic and intranuclear transfer of biologically active substances. While such vectors are highly effective, their use can produce problems in some applications. The use of antibodies or F(ab′)2 antibody fragments involves the production of high titers of these molecules with qualities which are compatible with therapeutic use. Further, the use of molecules with the size and complexity of antibodies can constitute a further disadvantage, in particular as regards use. U.S. Pat. No. 5,635,383 illustrates a further type of complex vector based on polylysine for transferring nucleic acids into cells.
The present application relates to novel polypeptides with advantageous properties both for transferring of substances into cells and as antiviral agents. The primary structure of these polypeptides is much simpler than antibodies and they are of reduced size. Further, preparation is easy and their potential applications are highly varied.
More particularly, the present invention stems from the discovery by the inventors that it is possible to identify, from whole antibodies, limited regions carrying a cellular penetration activity. The invention also stems from the discovery that it is possible to isolate, from whole antibodies, in particular from a single chain of these antibodies, peptides or polypeptides endowed with cell penetration activity. The present invention constitutes the first demonstration that a fragment of a single chain of an antibody can effectively penetrate into cells. The present invention also constitutes the first demonstration that such a fragment is also capable, advantageously, of transporting a substance of interest into said cell, and can preferably have an antiviral activity.